© 2003 Nature Publishing Group http://www.nature.com/naturebiotechnology
TECHNICAL REPORT
A genomics-guided
approach for discovering
and expressing cryptic
metabolic pathways
Emmanuel Zazopoulos1, Kexue Huang1,
Alfredo Staffa1, Wen Liu2, Brian O. Bachmann1,
Koichi Nonaka2, Joachim Ahlert2,3, Jon S. Thorson2,3,
Ben Shen2,4, and Chris M. Farnet1*
Published online 21 January 2003; doi:10.1038/nbt784
Genome analysis of actinomycetes has revealed the presence
of numerous cryptic gene clusters encoding putative natural
products1,2. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we
demonstrate the use of a high-throughput genome scanning
method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover
biosynthetic pathways encoding enediyne antitumor antibiotics
in a variety of actinomycetes. Comparative analysis of five
biosynthetic loci representative of the major structural classes
of enediynes reveals the presence of a conserved cassette of
five genes that includes a novel family of polyketide synthase
(PKS)3,4. The enediyne PKS (PKSE) is proposed to be involved
in the formation of the highly reactive chromophore ring structure (or “warhead”) found in all enediynes3,4. Genome scanning
analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective
growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be
much greater than previously thought. This technology can be
used to increase the scope and diversity of natural-product
discovery.
We have developed a high-throughput genome scanning method
to discover metabolic loci independently of their expression. This
approach takes advantage of the fact that the genes required for
secondary metabolite biosynthesis are typically clustered together
in a bacterial genome5. A shotgun DNA sequencing approach is
used to generate short (700 bp) random genome sequence tags
(GSTs) from a library of genomic DNA prepared from a microorganism. GSTs derived from genes that are likely to be involved in
the biosynthesis of natural products are identified by sequence
comparisons to a database of microbial gene clusters known to be
involved in natural-product biosynthesis. Selected GSTs are then
used to design screening probes to identify cloned subgenomic
fragments (for example, cosmids or bacterial artificial chromosomes (BACs)) containing the genes of interest as well as the
neighboring genes that together may constitute a biosynthetic
gene cluster (Fig. 1). Genome scanning provides an efficient way
1Ecopia
BioSciences, Inc., 7290 Frederick Banting, Montreal, Quebec H4S
2A1, Canada. 2Division of Pharmaceutical Sciences, 3Laboratory for
Biosynthetic Chemistry, and 4Department of Chemistry, University of
Wisconsin, Madison, WI 53706. *Corresponding author
(farnet@ecopiabio.com).
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to discover natural-product gene clusters because the analysis of a
relatively small number of GSTs provides reasonable assurance of
full genome representation. For example, analysis of 1,000 GSTs
from a genome of 8.5 Mbp (the approximate size of the genome of
an antibiotic-producing actinomycete) provides DNA sequence
sampling every 8.5 kbp (assuming random library coverage).
Given that natural-product gene clusters range in size from 20 to
200 kbp6,7 (C.F., unpublished data), it is expected that any given
gene cluster will be represented by anywhere from 2 to more than
20 of 1,000 GSTs analyzed. To date, we have used the genome scanning approach to successfully identify more than 450 naturalproduct gene clusters in a variety of actinomycetes (C.F., unpublished data).
We used the genome scanning method to isolate enediyne
biosynthesis genes from a variety of actinomycete strains known
to produce enediynes, a potent class of antitumor antibiotics8. The
enediynes induce irreversible DNA damage by a mechanism that
involves cycloaromatization of the warhead chromophore (Fig.
2A) to form highly reactive benzenoid diradicals that strip hydrogen atoms from the sugar phosphate backbone of the DNA helix9.
We chose the dynemicin and macromomycin biosynthesis gene
clusters to demonstrate the effectiveness of the genome scanning
method. Comparison with the C-1027 (ref. 3), calicheamicin4, and
neocarzinostatin (W. L., K.N., L. Nie, J. Bae, and B.S., unpublished
data) gene clusters reveals that the homology among all these loci
is limited to a set of five genes, including the gene encoding PKSE,
that form a putative “warhead gene cassette” (Fig. 2B). The conserved genes are generally arranged in a presumed operon with
Figure 1. A diagrammatic view of the genome scanning method for
high-throughput discovery of natural-product biosynthetic gene
clusters. Natural-product biosynthetic genes (in color) are clustered in
the bacterial genome (for simplicity, only a single gene cluster is
shown). High-molecular-weight genomic DNA is randomly fragmented
and small fragments are used to prepare a genome sampling library
(GSL) in a plasmid vector while large fragments are used to prepare a
cluster identification library (CIL) in a cosmid or BAC vector. Gene
sequence tags (GSTs) are generated from the GSL clones using a
universal primer located in the plasmid vector. The GSTs are compared
to a database of natural-product biosynthetic genes to identify tags
derived from genes involved in natural-product biosynthesis (“hot”
GSTs, colored inserts; step 1). These genes are then used as probes to
identify CIL clones containing the corresponding genes as well as their
neighboring genes (“hot” CIL clones). Overlapping CIL clones may be
identified by restriction fragment length mapping or during the
subsequent sequencing step. The hot CIL clones are randomly
fragmented and used to prepare a second plasmid library that provides
templates for sequencing (step 2). Sequencing and assembly of the
selected CIL clones result in a complete natural-product gene cluster
that is then annotated and entered into the database (step 3).
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A
D
We compared the other warhead cassette proteins to protein
007A
sequences present in the
GenBank nonredundant data009C
base to assess putative functions.
2 R , R = sugars
1
028D
One protein family (TEBC) is
similar to the 4-hydroxybenzoyl054A
CoA thioesterase of Pseudomonas
sp. strain CBS-3 in regions of the
059A
protein that have been shown to
132H
have an important role in catalysis11 and thus may be involved in
135E
polyketide chain release, cycliza145B
tion, or both (see Supplementary
3
4
Fig. 3 online). Three families of
E
B
unknown
proteins
(UNBL,
046E
DYNE
UNBV, and UNBU) show no significant homology to proteins in
CALI
100B
the public databases and therefore represent novel protein famMACR
171B
ilies that appear to be specific to
NEOC
enediyne
biosynthetic
loci.
Structural analysis of the UNBV
C-1027
PKSE TEBC UNBL UNBV UNBU
UNBU
proteins predicts that they are
secreted proteins with N-termiC
ACP
nal signal sequences, whereas the
NH
KS
?
KR
DH
? PPTE COOH
AT
UNBU proteins are predicted to
be integral membrane proteins
with seven or eight putative
Figure 2. Chemical structures of enediynes and genes involved in warhead formation. (A) The structures of
membrane-spanning
alpha
the enediynes dynemicin (1), calicheamicin (2), neocarzinostatin (3), and C-1027 (4). The common
cyclododecylpolyene skeleton found in all warheads is highlighted in red. The complete structure of
helices (see Supplementary Figs.
macromomycin has yet to be elucidated; however, the limited structural information available is consistent
4–6 online). Although the funcwith a chromophore ring system similar to that found in C-1027 (ref. 20). (B) Organization of the warhead
tions of the TEBC, UNBL, UNBV,
gene cassettes found in the dynemicin (DYNE), calicheamicin (CALI), macromomycin (MACR),
and UNBU proteins remain
neocarzinostatin (NEOC), and C-1027 loci. (C) Domain organization of the warhead PKS, consisting of KS
(ketoacyl synthase), AT (acyl transferase), ACP (acyl carrier protein), KR (keto reductase), DH
unknown, their strict association
(dehydratase), and PPTE (4′-phosphopantetheinyl transferase). (D) Organization of the warhead cassette
with the warhead PKS and their
genes found in loci from actinomycete strains not previously reported to produce enediyne natural products.
presence in all enediyne biosyn(E) Warhead cassette genes from actinomycete strains newly isolated from soil samples. 007A, locus found
thetic loci strongly suggest that
in Amycolatopsis orientalis; 009C, locus found in Streptomyces ghanaensis; 028D, locus found in
Kitasatosporia sp.; 054A, locus found in Micromonospora megalomicea subsp. nigra; 059A, locus found in
they have essential roles in the
Streptomyces cavourensis subsp. washingtonensis; 132H, locus found in Saccharothrix aerocolonigenes;
formation, stabilization, or trans135E, locus found in Streptomyces kaniharaensis; 145B, locus found in Streptomyces citricolor. Loci 046E,
port of the enediyne warhead.
100B, and 171B were found in new actinomycete isolates (Ecopia BioSciences Inc.).
We used the genome scanning
method to isolate natural-produnidirectional transcription and frequent overlap of translational
uct biosynthetic loci from a variety of actinomycete strains that
start and stop codons, suggesting that their gene products are
have been reported to produce various classes of natural products
coordinately expressed and functionally related. As these are the
but not enediyne compounds. Out of 50 actinomycete strains anaonly genes common to all enediyne loci analyzed to date, we prolyzed, eight (16%) were found to contain biosynthetic loci conpose that they constitute a functional unit responsible for the biotaining the enediyne warhead cassette (Fig. 2D), indicating that
genesis of the warhead, the single structural feature that is found
these strains could potentially produce enediyne natural products.
in all of the known enediynes9 (Fig. 2A).
This finding is surprising, as none of the eight strains was previThe PKSEs are likely to generate the carbon skeleton of the warously reported to produce enediynes, and it indicates that
head by catalyzing iterative cycles of acyl-coenzyme A (acyl-CoA)
enediyne biosynthetic loci occur at an unexpectedly high frequencondensation, ketoreduction and dehydration, using an acyl carricy in the actinomycetes. Enediyne loci occurred at a similar freer protein (ACP) domain as a covalent attachment site for the
quency in actinomycete strains newly isolated from soil samples: 3
growing carbon chain. The PKSEs contain enzymatic domains
out of 20 (15%) randomly selected strains were found to harbor
characteristic of known PKSs, including ketoacyl synthase (KS),
biosynthetic loci containing enediyne warhead cassette genes (Fig.
acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)
2E). It is notable that the strong conservation of the enediyne wardomains, as well as ACP domains3,4 (Fig. 2C and Supplementary
head gene sequence and gene order holds across several actinoFig. 1 online). Additional analysis of the PKSE sequences
mycete genera (Actinomadura, Amycolatopsis, Kitasatosporia,
described here further revealed a previously unidentified domain
Micromonospora, Saccharothrix, Streptomyces).
in the C-terminal region of the protein that is similar to 4′-phosFinally, we validated enediyne production by culturing strains
phopantetheinyl transferases10 (PPTases) and is likely to be
harboring warhead gene cassettes in a variety of fermentation
involved in post-translational autoactivation of the PKSE (Fig. 2C
media to test for activity in the biochemical induction assay (BIA),
and Supplementary Figs. 1, 2 online).
a modified prophage induction assay that detects agents that damHO
O
HO
H3CO
SSSCH3
OH
H
N
R 2O
OH
O
O
NH
O
O
H
N
H3C
HO
OR1
S
O
CH3
O
CH3
I
O
CH3
H3COOC
HOOC
OCH3
O
1
2
CH2
O
H3CO
© 2003 Nature Publishing Group http://www.nature.com/naturebiotechnology
N
H
O
O
O
CH3
O
O
O
O
O
O
H3C
O
O
OH
O
H3C
O
CH3HN
HO
O
O
(H3C)2N
HO
O
O
CH3 OH
OH
Cl
NH2
CH3
OH
2
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TECHNICAL REPORT
© 2003 Nature Publishing Group http://www.nature.com/naturebiotechnology
A
cates that additional classes of enediyne natural products remain
to be discovered.
The genome sequences of two actinomycetes, Streptomyces
coelicolor1 and Streptomyces avermitilis2, reveal several sets of
apparently cryptic natural-product gene clusters, suggesting that
these well-studied strains may produce a greater number of bioactive compounds than has been detected by fermentation broth
analysis16. We have demonstrated here that standard fermentation
broth screening failed to identify many bacterial strains that can
produce enediyne antibiotics. The ability of actinomycetes to produce antibiotics and other bioactive natural products has apparently been greatly underestimated. This work demonstrates the
utility of genome analysis in discovering hidden metabolic potential and directing rational approaches for the expression, detection, and purification of new bioactive natural products.
B
Experimental protocol
Figure 3. Enediyne production measured by the BIA. The presence of
inhibition zones surrounded by blue rings is indicative of β-galactosidase
induction due to DNA damage. At higher dilutions, only β-galactosidase
activity is observed, as the DNA damaging activity of the enediynes
occurs at concentrations that are not bactericidal12. (A) BIA activity from
microorganisms producing calicheamicin, dynemicin, macromomycin,
and neocarzinostatin and (B) from Amycolatopsis orientalis (007),
Streptomyces ghanaensis (009), and Streptomyces citricolor (145),
organisms not previously reported to produce enediynes, as well as from
two new actinomycete isolates, 046 and 171. Media AA, YA, and ZA do
not support enediyne production.
age DNA and is commonly used to assay enediyne production12. In
most media these strains did not have detectable BIA activity.
However, all strains produced BIA activity when grown in specialized media selected for their ability to support enediyne production (Fig. 3). These results provide strong evidence that these
strains are able to produce enediyne natural products and that the
expression of enediyne biosynthetic loci is restricted to certain fermentation conditions.
Although all of the 11 loci encoding unknown enediynes contain the conserved warhead cassette genes, these loci differ considerably from one another. They also differ from the loci that
encode the structurally characterized enediynes in the surrounding genes, which probably generate the structural units appended
to the warhead chromophore (unpublished data). This may indicate that each locus potentially encodes a different enediyne natural product. Considering that a total of only 11 members of the
enediyne family have been described to date9, it is likely that some
of the unknown loci described here produce new classes of
enediynes. As the enediynes are the most potent antitumor agents
ever discovered8, the discovery of new classes holds great interest.
While they are too toxic for systemic use in unmodified form, the
enediynes have proven to be effective anticancer drugs when conjugated to polymers or antibodies. For example, a polymer-conjugated form of neocarzinostatin has been used clinically to treat
hepatoma in Japan since 199413, whereas a calicheamicin–antiCD33 antibody conjugate (Mylotarg) was approved in the United
States in 2000 for the treatment of acute myelogenous leukemia14.
In addition, several C-1027–antibody conjugates are currently
under clinical evaluation as anticancer drugs15. This work indiwww.nature.com/naturebiotechnology
•
Genome scanning. The genomes of the dynemicin-producing organism
(Micromonospora chersina strain M956-1, ATCC 53710) and the macromomycin-producing organism (Streptomyces macromyceticus strain M480M1, NRRL B-5335) were analyzed by genome scanning (described in
patent application CA 2,352,451). Briefly, high-molecular-weight genomic
DNA was prepared from each organism17 and used to generate a smallinsert genomic sampling library (GSL) and a large-insert cluster identification library (CIL). Both libraries contain randomly fragmented genomic
DNA and therefore are representative of the entire genome. For the generation of the GSL, genomic DNA was sonicated and fragments of 1.5–3 kbp
were prepared by agarose gel electrophoresis and cloned into plasmid vectors. For the generation of the CIL, genomic DNA was fragmented to a size
range of 30–50 kbp by partial digestion with the restriction endonuclease
Sau3A1 before being cloned into cosmid vectors. One thousand gene
sequence tags (GSTs) (average read length, 700 bp) were obtained from
each GSL, translated into amino acid sequence, and compared to a proprietary database of microbial natural-product biosynthetic loci (DECIPHER
Database, Ecopia BioSciences Inc., Montreal, Canada; http://www.ecopiabio.com) using the basic local alignment search tool protein database
(BLASTP) software (http://www.ncbi.nlm.nih.gov/) to identify gene
sequences likely to be involved in the production of natural products. The
efficiency of the genome scanning method depends in part on the ability to
distinguish genes involved in natural-product biosynthesis from those
involved in primary metabolism, and thus will vary according to the size
and breadth of the database used for comparison. The probability that a
particular gene cluster will be identified by analysis of a given number of
GSTs is improved if the database contains a large number of gene clusters
representing a broad range of natural-product classes. The DECIPHER
database was initially populated with gene clusters representing a diverse
range of natural product classes collected from public databases such as
GenBank, and subsequently enriched with gene clusters discovered at
Ecopia. Selected gene sequences were used to design screening probes to
identify cosmids containing putative natural-product gene clusters from
the CIL. Selected cosmids were sequenced by the shotgun method, and
overlapping cosmids were identified by using the cosmid end sequences as
probes to screen the CILs.
Genome scanning was also used to isolate natural-product biosynthetic
loci from 50 previously isolated actinomycete strains as well as from 20
new actinomycete strains isolated from soil samples. Actinomycete strains
used to isolate natural-product biosynthetic loci include Amycolatopsis orientalis ATCC 43491 (vancomycin producer), Streptomyces ghanaensis
NRRL B-12104 (moenomycin producer), Kitasatosporia sp. CECT 4991
(taxane producer), Micromonospora megalomicea subsp. nigra NRRL 3275
(megalomicin producer), Streptomyces cavourensis subsp. washingtonensis
NRRL B-8030 (chromomycin producer), Saccharothrix aerocolonigenes
ATCC 39243 (rebeccamycin producer), Streptomyces kaniharaensis ATCC
21070 (coformycin producer), Streptomyces citricolor IFO 13005 (aristeromycin and neplanocin A producer). Enediyne biosynthetic loci were
identified by the presence of the conserved enediyne warhead cassette
genes as well as other genes frequently found in biosynthetic loci encoding
other natural-product classes (data not shown). The neocarzinostatin
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locus was cloned from Streptomyces carzinostaticus subsp. neocarzinostaticus ATCC 15944, sequenced by the shotgun method, and confirmed to
direct neocarzinostatin biosynthesis by gene inactivation and complementation experiments (W. L., K.N., L. Nie, J. Bae, and B.S., unpublished
data).
Protein homology analysis. To identify the PKSE PPTase domain, the Cterminal regions of the PKSEs from the neocarzinostatin, calicheamicin
and macromomycin biosynthetic loci were analyzed for their folding using
secondary structure predictions and solvation potential information18.
Comparison searches using a database of known three-dimensional structures of proteins revealed similarities with Sfp, the 4′-phosphopantetheinyl
transferase from the Bacillus subtilis surfactin biosynthetic locus19 (PDB id:
1QR0). Protein alignments based on secondary structure predictions as
well as identification of conserved amino acids important for cofactor
binding can be found in the Supplementary Figure 2 online. Amino acid
sequence alignments of the PKSE, TEBC, UNBL, UNBV, and UNBU proteins from the calicheamicin, macromomycin, dynemicin, C-1027, and
neocarzinostatin biosynthetic loci can also be found in the Supplementary
Figures 1 and 3–6. Where applicable, putative functions for these proteins
were assessed by comparison to protein sequences present in the GenBank
nonredundant database using the BLASTP software and by subcellular
protein localization prediction using the PSORT program available at
http://psort.nibb.ac.jp./
Fermentation and activity screening. Organisms were initially grown in
25 ml of TSB17 seed medium for 60 h at 28 °C and then diluted 30-fold in
25 ml production medium. Production medium for calicheamicin was
composed of 20 g of sucrose, 2 g of Bactopeptone (Becton Dickenson,
Sparks, MD), 5 g of cane molasses, 0.1 g of FeSO4•7H2O, 0.2 g of
MgSO4•7H2O, 0.5 g of KI, and 5 g of CaCO3 per liter. Production medium
for macromomycin was composed of 40 g of glucose, 5 g of dried yeast, 1 g
of K2HPO4, 1 g of MgSO4, 1 g of NaCl, 2 g of (NH4)2SO4, 2 g of CaCO3, 1
mg of FeSO4•7H2O, 1 mg of MnCl2•4H2O, 1 mg of ZnSO4•7H2O, and 0.5
mg of NaI per liter. Production medium for dynemicin was composed of
10 g of corn starch, 5 g of Pharmamedia (Southern Cotton Oil Co.,
Memphis, TN), 1 g of CaCO3, 0.05 g of CuSO4•5H2O, and 0.5 mg of NaI
per liter. Production medium for neocarzinostatin was composed of 40 g
of glucose, 15 g of casamino acids, 5 g of NaCl, 2 g of CaCO3, 1 g of
K2HPO4 and 12.5 g of MgSO4 per liter. Selective media supporting
enediyne production in organisms not previously reported to express
enediyne compounds and new actinomycete isolates were as follows. For
A. orientalis (007), medium was as described for calicheamicin production. For S. ghanaensis (009), production medium was composed of 30 g
of glycerol, 15 g of distiller’s solubles, 10 g of Pharmamedia, 10 g of fish
meal, and 6 g of CaCO3 per liter. For S. aerocolonigenes (132), S. kaniharaensis (135), and Ecopia strain 171, production media were composed
of 60 g of molasses, 20 g of soluble starch, 20 g of fish meal, 0.1 g of
CuSO4•5H2O, 0.5 mg of NaI, and 2 g of CaCO3 per liter. For S. citricolor
(145) and Ecopia strain 046, the production medium was composed of 10
g of glucose, 10 g of starch, 15 g of soybean meal, 1 g of K2HPO4, 3 g of
NaCl, 1 g of MgSO4•7H2O, 7 mg of CuSO4•5H2O, 1 mg of FeSO4•7H2O, 8
mg of MnCl2•4H2O, and 2 mg of ZnSO4•5H2O per liter. For S. cavourensis
subsp. washingtonensis (059), production medium was composed of 20 g
of glucose, 5 g of Bactopeptone, 5 g of beef extract, 5 g of NaCl, 3 g of yeast
extract, and 2 g of CaCO3 per liter. Examples of media not supporting
enediyne production include media AA (10 g of glucose, 40 g of corn dextrin, 15 g of sucrose, 10 g of casein hydrolysate, 1 g of MgSO4•7H2O, and 2
g of CaCO3 per liter) and CECT media 32 and 131 (Colección Española de
Cultivos Tipo, Valencia, Spain) herein referred to as media YA and ZA,
respectively.
Production cultures (25 ml) were incubated for 7 d at 28 °C under constant agitation. Culture (2 ml) was removed and clarified by centrifugation to provide supernatant samples. The rest of the culture (supernatant
and mycelia) was extracted with an equal volume of methanol under agitation for 30 min. Extracts were clarified by centrifugation and diluted
accordingly in their respective media supplemented with 50% methanol.
The BIA was performed as described12. Briefly, 10 µl of supernatant or
extract and twofold serial dilutions thereof were applied to agar plates
seeded with Escherichia coli BR513 and incubated for 3 h at 37 °C. Soft
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agar containing 0.7 mg/ml of X-gal was added onto the plate and color
development was observed within 30 min. BIA activity was tested on serial dilutions of methanol extracts for microorganisms producing
calicheamicin and dynemicin as well as for microorganisms 046, 145, and
171. Serial dilutions of supernatants from microorganisms producing
macromomycin and neocarzinostatin as well as from microorganisms 007
and 009 were assayed for BIA activity. All production media used in this
study were assayed alone and shown to be negative for BIA activity (data
not shown).
GenBank accession numbers. The DNA and protein sequences described
here are deposited in GenBank under accession numbers AF548580,
AF548581, and AF546139–AF546157.
Note: Supplementary
Biotechnology website.
information
is
available
on
the
Nature
Acknowledgments
We thank S. Mercure, V. Dodelet, and M. Piraee for helpful discussions and
J. McAlpine for critical reading of the manuscript. B.S. is a recipient of a NSF
CAREER Award (MCB9733938) and a NIH Independent Scientist Award
(AI51689). Enediyne studies in the Shen lab are supported in part by NIH
grant CA78747. Research in the Thorson lab is supported in part by NIH
grants CA84347, GM58196, and AI52218. J.S.T. is an Alfred P. Sloan Fellow.
Competing interests statement
The authors declare that they have no competing financial interests.
Received 21 October 2002; accepted 4 December 2002
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